Ultrastructural analysis of lysosome reactions to inside-out cell membrane vesicles in a cell-free system

Abstract

Summary: Lysosome reactions were ultrastructurally analyzed using a cell-free system with inside-out cell membrane vesicles (IOVs) prepared from rat erythrocyte ghosts in an alkaline buffer and with wheat germ agglutinin-coated colloidal gold particles (WGA-CGs). The submembranous surface coat in the ghosts was depleted from the IOVs' outer surfaces. When lysosomes from rat liver were incubated with these IOVs, some of the trilaminar membranes of the lysosomes and IOVs came into close contact and formed a five-laminar structure without an intermembranous gap. In other reactions, the membranes of both structures formed one continuous trilaminar membrane along the margin of contact and ruffling fivelaminar structures in other regions. Several lysosomes exhibited invaginating hollows or projections that entrapped or encircled the IOVs. Similar five-laminar structures were seen at a few points of contact between the IOVs and the hollowing or projecting membranes. In contrast, such reactions were much rarer when IOVs with reconstituted spectrins and actins on their outer surface were used. The formation of tubuliform pits with membrane-bound WGA-CGs was also observed after the incubation of lysosomes with WGA-CGs. These observations suggest that lysosomes fuse with cytoskeleton-depleted IOVs, wrap arm-like projections around them, enclose them by invagination or incorporate their membrane-bound macromolecules through the process of tubuliform invagination. Furthermore, the fusion and wrapping processes are not necessarily independent.Abbreviations: anti-Sp-IM, anti-rat erythrocyte spectrin IgG-coated IM; BSA-CG, bovine serum albumin-coated CG; buffer 1, KCl-sucrose-10 mM HEPES buffer (pH 7.0); CG, colloidal gold particle; Fer-WGA, ferritin-labeled WGA; GlcNAc, N-acetyl-D-glucosamine; IM, iron magnetic particle; IOV, inside-out vesicle; ROV, right-side-out vesicle; SpA-IOV, spectrin-and actinreconstituted IOV; TEM, transmission electron microscopy; WGA, wheat germ agglutinin; WGA-CG, WGA-coated CG.